ABSTRACT Yellow fever virus (YFV) represents a mosquito-borne emerging/re-emerging human pathogen that causes 20- 50% mortality and is endemic in >40 countries. The current live attenuated YFV vaccine was developed in 1936 and following the establishment of a virus seed lot system, it has not been modified or otherwise improved in over 50 years. According to the CDC, this vaccine causes 47 serious adverse events (SAE) per million vaccinations (SAE defined as resulting in hospitalization, long-term disability, or death). Some reports indicate that vaccine-associated neurological disease may occur at a rate of up to 1 case per 10,000 vaccinations and YFV vaccination of infants <9 months of age has been contraindicated since the 1960's due to high rates of vaccine-associated encephalitis in this age group. More recently, YFV vaccination has been found to cause severe viscerotropic disease in a substantial number of patients >60 years of age (an incidence rate of approximately 1:50,000 doses administered) and these cases result in approximately 50% mortality. This indicates that YFV vaccination is not only contraindicated in infants, but is also not recommended in the elderly due to the increased risk of severe and life-threatening disease. Increased monitoring efforts have also documented several cases of vaccine-related fatalities in young, otherwise healthy adults with no known pre- existing immune deficiencies. The overall (all ages) mortality rate following YFV vaccination is estimated at 1 to 2 deaths per million doses ? but there is currently no alternative to live YFV vaccination. In this proposal, we will prepare an inactivated YFV vaccine under GMP conditions and perform the necessary safety, potency, and stability studies required for a future IND submission to the FDA. This vaccine is based on proprietary new technology used to develop inactivated vaccine formulations that can be administered to vulnerable populations such as infants and the elderly, in addition to other healthy populations. Preliminary data is provided that demonstrates an H2O2-based inactivated YFV vaccine is feasible to manufacture, highly immunogenic, and provides full protective immunity against lethal viscerotropic yellow fever. In this project, we will prepare clinical grade vaccine under cGMP conditions, perform in vitro and in vivo safety/toxicity tests, and determine vaccine potency and long-term stability. The successful completion of these objectives will result in cGMP-grade vaccine material suitable for future initiation of a Phase I clinical trial.